Method of measuring homocysteine

ABSTRACT

A method for detecting or measuring homocysteine in a sample includes the steps of (a) reacting a D-amino acid present in a sample with a D-amino acid converting enzyme to convert the D-amino acid into a substance that does not serve as a substrate of D-amino acid oxidase or D-amino acid acetyltransferase; (b) reducing homocysteine in the sample with a thiol compound; (c) reacting the reduced homocysteine with a methyltransferase and a methyl donor to newly produce D-amino acid; and (d) reacting the produced D-amino acid with the D-amino acid oxidase or the D-amino acid acetyltransferase in the presence of an SH reagent to produce hydrogen peroxide, and color-developing the produced hydrogen peroxide by using an oxidative color-developing agent.

TECHNICAL FIELD

The present invention relates to a method of detecting or measuringhomocysteine in a sample. More specifically, the present inventionrelates to a method of measuring homocysteine including the step ofremoving a D-amino acid present in a sample in advance.

BACKGROUND ART

As a method of measuring homocysteine, a method of reacting homocysteinein a sample with homocysteine methyltransferase and D-methioninemethylsulfonium, and then detecting the produced D-methionine withD-amino acid oxidase has been reported (see WO 02/02802). However, it isknown that biological samples contain D-amino acids such as D-alanineand D-serine in small amounts, and these amino acids increase in thecase of renal diseases or the like (e.g., see Fukushima, T., Biol.Pharm. Bull., 1995, Vol. 18, No. 8, pp. 1130–1132). It is believed thatD-alanine and D-serine, which can be a substrate of D-amino acidoxidase, leads to a positive reading in the method of measuringhomocysteine. Therefore, as described in WO 02/02802, in order to avoidthe influence of endogenous D-amino acids that are originally present ina sample, it is necessary to subtract a value obtained by measurement bythe same operation except that homocysteine methyltransferase is notcontained from a measured value in the case of containing this enzyme.That is to say, it is necessary to provide a sample blank for eachindividual sample to measure the amount of endogenous D-amino acids.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method ofmeasuring homocysteine that is not affected by endogenous D-amino acids,that is, that does not require sample blanks.

As a result of in-depth research in order to achieve the above object,it became possible to provide a method of measuring homocysteine that isnot affected by endogenous D-amino acids, that is, that does not requiresample blanks by leading D-alanine and/or D-serine to the outside of thereaction system of the homocysteine measurement by enzyme actions.

The present invention provides a method for detecting or measuringhomocysteine in a sample, and the method includes the steps of: (a)reacting a D-amino acid present in a sample with a D-amino acidconverting enzyme to convert the D-amino acid into a substance that doesnot serve as a substrate of D-amino acid oxidase or D-amino acidacetyltransferase; (b) reducing homocysteine in the sample with a thiolcompound; (c) reacting the reduced homocysteine with a methyltransferaseand a methyl donor to newly produce D-amino acid; and (d) reacting theproduced D-amino acid with the D-amino acid oxidase or the D-amino acidacetyltransferase in the presence of an SH reagent to produce hydrogenperoxide, and color-developing the produced hydrogen peroxide by usingan oxidative color-developing agent.

In a preferred embodiment, the step (a) is a step of reacting D-alaninepresent in a sample with D-alanyl-D-alanine ligase (which may bereferred to “Ddl” hereinafter) in a presence of adenosine triphosphateto convert the D-alanine into D-alanyl-D-alanine and/or a step ofreacting D-serine present in a sample with D-serine dehydratase (whichmay be referred to “Dsd” hereinafter) to convert the D-serine intopyruvic acid.

In a preferred embodiment, the methyltransferase is homocysteinemethyltransferase, and the methyl donor is D-methionine methylsulfonium.

In a preferred embodiment, in the step (d), the produced hydrogenperoxide is detected or measured by color-development using peroxidaseand an oxidative color-developing agent.

The present invention also provides a reagent kit for measuringhomocysteine comprising D-alanyl-D-alanine ligase and/or D-serinedehydratase; a thiol compound; methyltransferase; a methyl donor;D-amino acid oxidase or D-amino acid acetyltransferase; an SH reagent;and an oxidative color-developing agent.

The present invention further provides a method for detecting ormeasuring homocysteine in a sample, comprising the steps of reacting aD-amino acid present in a sample with a D-amino acid converting enzymeto convert the D-amino acid into a substance that does not serve as asubstrate of D-amino acid oxidase or D-amino acid acetyltransferase.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram of the reaction in a method of measuringhomocysteine using homocysteine transferase and D-methioninemethylsulfonium.

FIG. 2 is a schematic diagram showing construction of expression vectorpKdlA.

FIG. 3 is a schematic diagram showing construction of expression vectorpKdlB.

FIG. 4 is a graph showing a relationship between the concentration ofD-alanyl-D-alanine ligase and the sensitivity of the homocysteinemeasurement.

FIG. 5 is a graph showing a relationship between the concentration ofD-serine dehydratase and the sensitivity of the homocysteinemeasurement.

FIGS. 6( a)–6(c) are graphs showing a correlation between theconcentration of the measured homocysteine according to (a) aconventional single channel method, (b) a conventional double channelmethod, and (c) the method of the present invention and theconcentration measured by a HPLC method.

DETAILED DESCRIPTION OF THE INVENTION

Principle of Homocysteine Measurement:

The method of measuring homocysteine of the present invention is basedon the principle that homocysteine in a sample is subjected to areduction treatment with a thiol compound, and is reacted with amethyltransferase in the presence of a methyl donor (first process), andthen the produced D-amino acid or D-amino acid derivative is measured(second process). For example, as shown in FIG. 1, when D-methionineproduced in the first process is reacted with D-amino acid oxidase inthe second process, hydrogen peroxide is produced, and the hydrogenperoxide can be led to an oxidative color-developing agent commonly usedin the presence of an SH reagent so as to be determinedcolorimetrically. When the D-methionine is reacted with D-amino acidacetyltransferase, the produced coenzyme A is led to hydrogen peroxideusing acyl coenzyme A synthetase [EC 6.2.1.3] and acyl coenzyme Aoxidase [EC 1.3.3.6], and this can be determined in the same manner.

The method of detecting or measuring homocysteine of the presentinvention is characterized in that in order to eliminate the influenceof endogenous D-amino acids when measuring D-amino acids, first, aD-amino acid present in a sample is reacted with a D-amino acidconverting enzyme to convert the D-amino acid to a substance that doesnot serve as a substrate of D-amino acid oxidase or D-amino acidacetyltransferase.

More specifically, the method of detecting or measuring homocysteine ofthe present invention includes the steps of:

(a) reacting a D-amino acid present in a sample with a D-amino acidconverting enzyme to convert the D-amino acid to a substance that doesnot serve as a substrate of D-amino acid oxidase or D-amino acidacetyltransferase;

(b) reducing homocysteine in the sample with a thiol compound;

(c) reacting the reduced homocysteine with a methyltransferase and amethyl donor to newly produce a D-amino acid; and

(d) reacting the produced D-amino acid with the D-amino acid oxidase orD-amino acid acetyltransferase in the presence of an SH reagent toproduce hydrogen peroxide, and color-developing the produced hydrogenperoxide by using an oxidative color-developing agent.

Any sample can be used as a sample to be subjected to detect or measurehomocysteine by the method of the present invention, as long as it isbelieved to contain homocysteine. The homocysteine can be present in theform of, not only reduced homocysteine, but also oxidized homocysteinethat is bound to another molecule by a disulfide bond such as a complexwith a protein, a homocysteine dimer and a homocysteine-cysteine dimer.For example, serum, plasma, blood, urine and a dilution thereof can beused.

Step (a):

As the D-amino acid converting enzyme that is used in the method of thepresent invention, any D-amino acid converting enzyme can be used, aslong as it can react with a D-amino acid to convert the D-amino acidinto a substance that does not serve as a substrate of D-amino acidoxidase or D-amino-acid acetyltransferase so as to lead it to theoutside of the reaction system of the homocysteine measurement. In thepresent invention, a D-amino acid converting enzyme that can react withD-alanine and/or D-serine is preferable. As an enzyme that reacts withD-alanine, D-alanyl-D-alanine ligase [EC 6.3.2.4], D-alaninehydroxylmethyltransferase [EC 2.1.2.7], D-alanine-y-glutamyl-transferase[EC 2.3.2.14], or the like can be used. As an enzyme that reacts withD-serine, D-serine dehydratase [EC 4.3.1.18], diaminopropionateammonia-lyase [EC 4.3.1.18], or the like can be used. These enzymes canbe used alone or in combination if necessary. As the enzyme that reactswith D-alanine, D-alanyl-D-alanine ligase is preferably used, and as theenzyme that reacts with D-serine, D-serine dehydratase is preferablyused.

Any D-alanyl-D-alanine ligase (Ddl) derived from any sources can be usedin the method of the present invention, enzymes, as long as it cancondense two D-alanine molecules to produce D-alanyl-D-alanine. Forexample, Ddls derived from almost all bacteria can be utilized. Enzymesderived from E. coli are preferable. “E. coli” in this specificationrefers to Escherichia coli described in Bergey's Manual of DeterminativeBacteriology, the 8th edition (edited by R. E. Buchanan and N. E.Gibbons, The Williams & Wilkins Company, Baltimore, pp. 295–296, 1974)and its variants and modified forms.

As Ddl used in the method of the present invention, enzymes derived fromE. coli having an amino acid sequence estimated from the base sequenceof GenBank Accession No. J05319 described in Biochemistry 30:1673–1682(1991) or GenBank Accession No. AE000118 REGION: 18688..19608 describedin Journal of Bacteriology 167: 809–817 (1986) are preferably used.Furthermore, enzymes derived from microorganisms belonging to the genusof Bacillus, Enterococcus, Lactobacillus and the like also can be used.The sequence can be modified in some amino acids (e.g., addition,deletion or substitution of one or more amino acids), as long as the Ddlactivity does not disappear. The Ddl can be obtained by any methodswell-known to those skilled in the art, for example, by a method ofpreparing a crude enzyme from the E. coli (e.g., an E. coli strain thathas been transformed by introducing ddl gene obtained from bacteria orthe like) and then purifying it by, for example, various chromatographytechniques.

The D-serine dehydratase (Dsd) used in the method of the presentinvention is an enzyme also called D-serine ammonia-lyase, D-serinedehydrase, D-hydroxyamino acid dehydratase, D-serine hydrase, orD-serine deaminase. Dsd derived from any sources can be used, as long asit can deaminate D-serine to produce pyruvic acid. Enzymes derived fromE. coli having an amino acid sequence estimated from the base sequenceof GenBank Accession No. J01603 described in J. Bacteriol. 154(3),1508–1512 (1983) are preferably used. Furthermore, Dsds derived frommicroorganisms belonging to the genus of Pseudomonas, Bacillus,Salmonella, Fusobacterium, Vibrio, Shigella, Ralstonia and the like canbe used. The sequence can be modified in some amino acids (e.g.,addition, deletion or substitution of one or more amino acids), as longas the Dsd activity does not disappear. The Dsd can be obtained by anymethods well-known to those skilled in the art, for example, by a methodof preparing a crude enzyme from E. coli (e.g., an E. coli strain thathas been transformed by introducing dsd gene obtained from bacteria orthe like) and then purifying it by, for example, various chromatographytechniques.

The ddl gene and the dsd gene can be obtained by a method commonly usedby those skilled in the art, based on the estimated amino acid sequencesdescribed above. For example, methods performing plaque hybridization,colony hybridization, PCR or the like can be employed, where a part orall of the genes encoding Ddl or Dsd or genes containing these sequencesare used as probes. The gene source of Ddl or Dsd is not limited to E.coli, but other species of bacteria can be used as well.

In this specification, “ddl gene” refers to a DNA chain or DNA sequencethat encodes a polypeptide having the Ddl activity that exhibits thecharacteristics of Ddl, and “dsd gene” refers to a DNA chain or DNAsequence that encodes a polypeptide having the Dsd activity thatexhibits the characteristics of Dsd. In both cases, a polypeptide havinga modification (e.g., addition, deletion or substitution) in the aminoacid sequence that does not affect the activity of the enzyme asdescribed above may be encoded. Moreover, a plurality of sequences mayencode the same polypeptide, for example, due to degeneracy.Furthermore, the ddl gene or the dsd gene may be derived from naturalsources or may be totally synthesized or semi-synthesized chemically.

The obtained ddl gene or the dsd gene is, for example, ligated to anexpression vector that can multiplicate and is introduced into a hostsuch as E. coli. The expression vector used herein may be any expressionvector, as long as it can be usually used for E. coli, and for example,ColE1, pCR1, pBR322, pMB9 and the like can be preferably used.

In order to express a large amount of DNA encoding Ddl or Dsd in E.coli, or to increase the expression amount thereof, a promoter forcontrolling transcription and translation may be incorporated into a 5′upstream region of the DNA chain of the vector, and/or a terminator maybe incorporated into a 3′ downstream region thereof. Such a promoterand/or a terminator may be derived from a ddl gene or dsd gene itself,derived from a gene that is well known such as β-galactosidase gene, orobtained by artificially modifying these known genes. Therefore,expression vectors in which such control sequences are incorporated arepreferably used as the expression vector, and examples thereof includepTrc99A, pKK223-3 (which are manufactured by Amersham PharmaciaBiotech), and pET3, pET-11 (which are manufactured by Stratagene),although there is no limitation to these vectors.

Any microorganisms can be used as a host for expressing Ddl or Dsd, butbacteria are preferable and E. coli is more preferable. A transformantfor expressing Ddl or Dsd can be produced by a method known in the fieldof genetic engineering, for example, by a rubidium chloride method (J.Mol. Biol., 166:557, 1983). The thus obtained transformant of E. coliwhose ability of expressing Ddl or Dsd is increased is cultured so thatDdl or Dsd can be obtained.

The Ddl or Dsd obtained in the above-described manner may be used aloneor in combination.

When Ddl is applied to a reagent for measuring homocysteine, theproduced D-alanyl-D-alanine can not be a substrate of D-amino acidoxidase, so that it is possible to eliminate the D-alanine contained ina sample. There is no limitation regarding the amount of enzyme used, aslong as it has a concentration that can eliminate the D-alanine in thesample, and for example, the amount can be 0.01 to 100 U/mL, andpreferably is 0.1 to 10 U/mL. In the present invention, the amount ofDdl that produces 1 μmol of D-alanyl-D-alanine per minute at 37° C.,using D-alanine as the substrate is defined as 1 unit. There is also nolimitation regarding the amounts of ATP and Mg ions to be used that isnecessary to exhibit the activities, as long as they have aconcentration that can eliminate D-alanine, and for example, for ATP, anamount of 0.1 to 10 mM, and for Mg ions, an amount of 0.1 to 20 mM arepreferable. This treatment of a sample with enzymes can be performedalone, or can be performed simultaneously with the following steps (b)and (c).

When Dsd is used as a reagent for measuring homocysteine, D-serine canbe removed from the reaction system by converting D-serine contained ina sample into pyruvic acid. There is no limitation regarding the amountof enzyme used, as long as it has a concentration that can eliminateD-serine in the sample, and for example, the amount can be 0.001 to 10U/mL, and preferably is 0.01 to 1 U/mL. In the present invention, theamount of Dsd that degrades 1 μmol of D-serine per minute at 37° C. isdefined as 1 unit. This treatment of a sample with enzymes can beperformed alone, or can be performed simultaneously with the followingsteps (b) and (c).

Step (b):

The step (b) in the method of the present invention is a step ofreducing homocysteine in various forms in a sample with a thiol compoundinto a reduced homocysteine.

The thiol compound used in the method of present invention is notparticularly limited, and includes, for example, dithiothreitol,mercaptoethanol, N-acetylcysteine, dithioerythritol, and thioglycolicacid. Any concentration of the thiol compound may be employed as theconcentration of the thiol compound, as long as it is in a range thatallows oxidized homocysteine to reduced homocysteine. Preferably, theconcentration is 0.1 mM or more in terms of thiol groups, and morepreferably 1 mM or more.

Step (c):

The step (c) in the method of the present invention is a step ofreacting the reduced homocysteine made in the step (b) above with amethyltransferase and a methyl donor so as to newly produce D-aminoacid. In the present invention, a methyltransferase using homocysteineas a methyl acceptor is preferably used, and D-methioninemethylsulfonium is preferably used as a methyl donor. That is, thereduced homocysteine in the sample is reacted with a methyltransferaseand D-methionine methylsulfonium to produce D-methionine.

There is no limitation regarding the methyltransferase, as long as itreacts with D-methionine methylsulfonium to catalyze the production ofD-methionine. Examples of the methyltransferase include homocysteinemethyltransferase [EC 2.1.1.10], 5-methyltetrahydrofolicacid-homocysteine S-methyltransferase [EC 2.1.1.13], and5-methyl-tetrahydropteroyltriglutamic acid-homocysteineS-methyltransferase [EC 2.1.1.14]. Preferably, homocysteinemethyltransferase [EC 2.1.1.10] can be used. This enzyme producesD-methionine, using D-methionine methylsulfonium as a methyl donor, inspite of its low specificity, as reported by G. Grue-Sorensen et al (J.Chem. Soc. Perkin Trans. I 1091-7 (1984)). The homocysteinemethyltransferase to be used may be derived from any sources, as long asit uses D-methionine methylsulfonium as a methyl donor. For example,enzymes derived from bacteria, yeasts, rats or the like can be used. Inthe present invention, the amount of the homocysteine methyltransferasethat produces 1 μmol of D-methionine per minute at 37° C., usinghomocysteine and D-methionine methylsulfonium as the substrates, isdefined as 1 unit.

Step (d):

The step (d) in the method of the present invention is a step ofreacting the D-amino acid produced in the step (c) with D-amino acidoxidase or D-amino acid acetyltransferase in the presence of an SHreagent to produce hydrogen peroxide, and color-developing the producedhydrogen peroxide by using an oxidative color-developing agent.

When D-amino acid is reacted with D-amino acid oxidase [EC 1.4.3.3],hydrogen peroxide is produced. This is led to an oxidativecolor-developing agent commonly used in the presence of an SH reagent soas to be determined calorimetrically. The D-amino acid oxidase may bederived from any sources. For example, it may be derived from animalorgans, bacteria, or fungi. Preferably, those derived from porcinekidney can be used. In the present invention, the amount of the D-aminoacid oxidase that converts 1 μmol of D-alanine into pyruvic acid perminute at 37° C. is defined as 1 unit.

When D-amino acid is reacted with D-amino acid acetyltransferase [EC2.3.1.36], coenzyme A is produced. Coenzyme A is reacted withacyl-coenzyme A synthetase [EC 6.2.1.3] and acyl-coenzyme A oxidase [EC1.3.3.6], sequencially, then hydrogen peroxide is produced. The producedhydrogen peroxide can be quantitatively determined in the same manner.The D-amino acid acetyltransferase may be derived from any sources. Forexample, those derived from yeasts can be used.

Examples of the SH reagent include an oxidizing agent such as Ellman'sreagent, a mercaptido forming agent such as p-mercuri benzoic acid, andan alkylating agent such as iodoacetic acid and N-ethylmaleimide, asdescribed in Biochemistry Dictionary (3^(rd) edition, p. 182, TokyoKagaku Doujin, 1998). Preferably, an alkylating agent, and morepreferably a maleimide compound, and most preferably, N-ethylmaleimidecan be used.

The produced hydrogen peroxide can develop the color of an ordinaryoxidative color-developing agent with peroxidase. As the oxidativecolor-developing agent, various Trinder reagents can be used incombination with a coupler reagent. This method is called the Trindermethod, and is commonly used in the field of clinical chemical analysis,which is not described herein in detail. It is preferable to use4-aminoantipyrine as the coupler reagent and to use ADOS[N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline], DAOS[N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline], HDAOS[N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline], MAOS[N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline], TOOS[N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline] or the like as theTrinder reagent. Furthermore, a leuco-type color-developing agent suchas o-tolidine, o-dianisidine, DA-67[10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazinesodium, manufactured by Wako Pure Chemical Industries Ltd.], and TPM-PS[N,N,N′,N′,N″,N″-hexa(3-sulfopropyl)-4,4′,4″-triaminotriphenylmethanehexasodium salt, Dojindo Laboratories], which do not require the couplerreagent, can be used as well. In particular, DA-67 and TPM-PS have amole absorption coefficient larger than that of the Trinder reagent, sothat the determination can be performed with higher sensitivity.

Reagent kit for homocysteine measurement:

The present invention provides a reagent kit for homocysteinemeasurement including (a) D-alanyl-D-alanine ligase and/or D-serinehydratase, (b) a thiol compound, (c) a methyltransferase and a methyldonor, and (d) D-amino acid oxidase or D-amino acidacetyltransterase+acyl-coenzyme A synthetase+acyl-coenzyme A oxidase, anSH reagent, and an oxidative color-developing agent. In general, these(a) to (d) are provided separately, but (a) to (c) may be provided as areagent for measurement that is previously prepared by mixing in abuffer solution.

Hereinafter, the present invention will be described more specificallyby way of examples, but the present invention is not limited by thefollowing examples.

EXAMPLE 1 Preparation of Recombinant D-alanyl-D-alanine Ligase (A)(DdlA) Derived from E. coli

(1-1) Synthesis of Probes and Obtainment of ddlA Gene

Synthetic primers shown in SEQ ID NOS: 1 and 2, which include EcoRI andPstI recognition sites, respectively, were synthesized based on the basesequence information of the ddlA gene from E. coli encoding an enzymehaving DdlA activity (Biochemistry 30:1673–1682 (1991), GenBankAccession No. J05319). PCR was performed in a buffer solution (2 μl ofKOD DNA polymerase (manufactured by TOYOBO Co., Ltd.; hereinafter,referred to as “KOD”), 10 μl of 10×KOD buffer solution, 10 μl of dNTPmixture, 5 μl of DMSO and 5 μl of distilled water), using these primersin an amount of 3 nmol (100 pmol/μl, 30 μl) each, and using 2 μl ofchromosomal DNA of E. coli JM109 as a template to give 1.09 kb DNAcontaining the DdlA structural gene.

(1-2) Preparation of Plasmid Including ddlA Gene

The thus obtained ddlA gene was ligated to a SmaI fragment of E. colivector pUC19 having DNA replication origin of E. coli ColE1 andampicillin resistance gene, and introduced in E. coli JM109 by therubidium chloride method (J. Mol. Biol., 166:557, 1983) to give atransformant having a recombinant plasmid containing the ddlA gene. Allthe restriction enzymes used in the Examples were obtained from TAKARABIO INC.

Using the recombinant plasmid in the transformant, the base sequence wasdetermined by using 377 Automate Sequencing System (manufactured byPerkinElmer Co., Ltd.) based on the dideoxy terminator method usingfluorescent-labeled primers. The ddlA has a structural gene region with1095 bases, and encodes 364 amino acids, which was completely consistentwith the above-described base sequence information on the ddlA gene.

(1-3) Preparation of Expression Vector and Transformant Including ddlAGene

The above-described recombinant plasmid was digested with restrictionenzymes EcoRI and PstI to give a DNA fragment, and then the 1.09 kb DNAfragment containing the ddlA DNA was purified by agarose gelelectrophoresis. E. coli vector pKK233-3 (manufactured by AmershamPharmacia Biotech) having ampicillin resistance gene was digested withrestriction enzymes EcoRI and PstI, and ligated to the obtained 1.09 kbDNA fragment to give an expression vector pKdlA (see FIG. 2). Thisexpression vector pKdlA is induced to express DdlA byisopropyl-β-D-thiogalactopyranoside (hereinafter, referred to as“IPTG”).

The obtained expression vector pKdlA was introduced into E. coli JM109by the rubidium chloride method, and one in which DdlA is expressed wasselected to give a transformant JM109-ddlA-3.

(1-4) Confirmation of Activity of Expressed DdlA

The obtained transformant JM109-ddlA-3 was cultured in 3 ml of LB liquidmedium (1% yeast extract, 2% bactopeptone, and 2% glucose) containingampicillin at 37° C. for about four hours with shaking. A 0.3 ml aliquotwas added to 10 ml of LB liquid medium and cultured at 37° C. for threehours with shaking, and IPTG (TAKARA BIO INC.) was added thereto suchthat the final concentration became 1 mM, and was cultured further forfour hours. The culture was centrifuged at 8000 rpm for 15 minutes tocollect cells. Then, after the cells were washed with 1 ml of 100 mMBis-tris-HCl buffer solution (pH 7.4) once, they were suspended in asolubilzation solution (100 mM Bis-tris-HCl buffer solution (pH 7.4), 1mM EDTA, 5 mM MgCl₂, and 100 μg/ml lysozyme) in a volume ten times thatof the cells. The suspension was treated twice with an ultrasonicgenerator (UD-200 manufactured by TOMY SEIKO Co., Ltd.) at scale 1 for10 seconds to disrupt the cells. A supernatant was obtained bycentrifugation at 15000 rpm for 10 minutes, and this was used as asample for measuring the Ddl activity. As a control, a transformant ofE. coli JM109 strain with non-recombinant pKK223-3 that had been treatedin the same manner was used.

The enzyme activity was measured in the following manner. First, areagent for measuring the activity (100 μl of 20 mM D-alanine, 20 mMATP, 100 mM HEPES, 40 mM MgCl₂, and 50 μl of 40 mM KCl) was added to thedisrupted cell solution (50 μl), and allowed to react at 37° C. for onehour. Then, 2 μl of the reaction mixture was spotted onto a silica gelthin layer, and then developed using ethanol:25% aqueous ammonia=74:26(w/w) as an eluent in a closed vessel. After development, a ninhydrinsolution (0.1 M citric acid buffer solution saturated with n-butanol,which contains 0.2% of ninhydrin) was sprayed to detect the producedD-alanyl-D-alanine. The D-alanyl-D-alanine was produced in the disruptedrecombinant cell solution in a significantly larger amount compared tothe disrupted control cell solution.

(1-5) Preparation of DdlA

The recombinant E. coli obtained in the above steps (1–3), which hashigh production ability of DdlA, was inoculated in 200 ml of LB medium(1% yeast extract, 2% bactopeptone, and 2% glucose) containingampicillin and was cultured at 37° C. for 15 hours. Then, the culturewas inoculated to 1.8 L of LB medium containing ampicillin in a jarfermenter with a volume of 5 L, and cultured at 37° C. for 100 minuteswith aeration and stirring. To the culture, IPTG was added such that thefinal concentration became 1 mM, and cultured further for 4 hours. Theculture was centrifuged at 8000 rpm for 10 minutes to collect cells. Thecells were suspended in a buffer solution (20 mM Bis-tris-HCl buffersolution (pH 7.4), 1 mM EDTA, and 5 mM MgCl₂) in a volume nine timesthat of the cells. The suspension was treated with an ultrasonicgenerator (UD-200 manufactured by TOMY SEIKO CO., Ltd.) to disrupt thecells. Then, the suspension was centrifuged at 15000 rpm for 10 minutesto remove the debris and thus a crude enzyme solution was obtained.

Ammonium sulfate was added to the obtained crude enzyme solution so asto be 5% saturation under ice cooling with stirring, and thereafter wasallowed to stand for 30 minutes, and then was centrifuged at 14000 rpm.Ammonium sulfate was added to the obtained supernatant so as to be 45%saturation under ice cooling with stirring, and thereafter allowed tostand for 30 minutes, and then centrifuged at 14000 rpm. The obtainedprecipitate was dialyzed overnight against a buffer solution (20 mMBis-tris-HCl buffer solution (pH 7.4), 1 mM EDTA, and 5 mM MgCl₂).

Then, the dialyzed crude enzyme solution was purified by columnchromatography (adsorption: a buffer solution A (20 mM Bis-tris-HClbuffer solution (pH 7.4), 1 mM EDTA, and 5 mM MgCl₂), elution: a buffersolution A—0.0 to 0.6 M sodium chloride gradient) using Q-SEPHAROSE FF(manufactured by Amersham Pharmacia Biotech). The Ddl active fractionswere collected and further purified by gel filtration columnchromatography with SEPHACRYL S-100 (manufactured by Amersham PharmaciaBiotech). The obtained active fractions were subjected to SDS-polyacrylamide gel electrophoresis and stained by Coomassie brilliant blue, whichconfirmed that the DdlA was purified to substantially a single band.

EXAMPLE 2 Preparation of Recombinant D-alanyl-D-alanine Ligase (B)(DdlB) Derived from E. coli

(2-1) Synthesis of Probe and Acquisition of ddlB Gene

Synthetic primers shown in SEQ ID NOS: 3 and 4 were synthesized, basedon the base sequence information of the ddlB gene of E. coli encoding anenzyme having the DdlB activity (Journal of Biochemistry 167:809–817(1986), GenBank Accession No. AE000118 REGION: 18688..19608). PCR wasperformed in a buffer solution (2 μl of KOD, 10 μl of 10×KOD buffersolution, 10 μl of dNTP mixture, 5 μl of DMSO and 5 μl of distilledwater), using these primers in an amount of 3 nmol (100 pmol/μl, 30 μl)each, and using 2 μl of chromosome DNA of E. coli JM109 as a template togive 0.92 kb DNA containing the DdlB structural gene.

(2-2) Preparation of Plasmid Including ddlB Gene

The thus obtained ddlB gene was ligated to a SmaI fragment of an E. colivector pUC19 having DNA replication origin of E. coli ColE1 and anampicillin resistance gene, and introduced into E. coli JM109 by therubidium chloride method (J. Mol. Biol., 166:557, 1983) to give atransformant having a recombinant plasmid containing the ddlB gene.

Using the recombinant plasmid in the transformant, the base sequence wasdetermined by using 377 Automate Sequencing System (manufactured byPerkinElmer Co., Ltd.) based on the dideoxy terminator method usingfluorescent-labeled primers. The ddlB has a structural gene region with921 bases, and encodes 306 amino acids, which was completely consistentwith the base sequence information on the ddlB gene.

(2-3) Preparation of Expression Vector and Transformant Including ddlBGene

An E. coli vector pKK233-3 (manufactured by Amersham Pharmacia Biotech)was digested with restriction enzymes EcoRI, and blunt-ended using DNABlunting Kit (manufactured by TAKARA BIO INC.), then dephosphorylatedusing alkaline phosphatase (manufactured by TAKARA BIO INC.), and afragment of the DdlB structural gene obtained in (2-1) was ligatedthereto to give an expression vector pKdlB (see FIG. 3). This expressionvector pKdlB is induced to express DdlB by IPTG.

The obtained expression vector pKdlB was introduced into E. coli JM109by the rubidium chloride method, and one in which DdlB is expressed wasselected to give a transformant JM109-ddlB-1.

(2-4) Preparation of DdlB

The recombinant E. coli obtained in the above steps (2-3), which hashigh production capacity of DdlB, was inoculated in 200 ml of LB medium(1% yeast extract, 2% bactopeptone, and 2% glucose) containingampicillin and was preliminarily cultured at 37° C. for 15 hours. Theculture was inoculated to 1.8 L of LB medium containing ampicillin in ajar fermenter with a volume of 5 L, and cultured at 37° C. for 100minutes with aeration and stirring. To the culture, IPTG was added suchthat the final concentration became 1 mM and the culture was culturedfurther for 4 hours. The culture was centrifuged at 8000 rpm for 10minutes to collect the cells. The cells were suspended in a buffersolution (20 mM Bis-tris-HCl buffer solution (pH 7.2), 1 mM EDTA, and 5mM MgCl₂) in a volume nine times that of the cells. The suspension wastreated with an ultrasonic generator (UD-200 manufactured by TOMY SEIKOCO., Ltd.) to disrupt the cells. Then, the suspension was centrifuged at15000 rpm for 10 minutes to remove the debris and thus a crude enzymesolution was obtained.

Ammonium sulfate was added to the obtained crude enzyme solution so asto be 25% saturation under ice cooling with stirring, and thereafter wasallowed to stand for 30 minutes, and then centrifuged at 14000 rpm.Ammonium sulfate was added to the obtained supernatant so as to be 50%saturation under ice cooling with stirring, and thereafter allowed tostand for 30 minutes, and then centrifuged at 14000 rpm. The obtainedprecipitate was dialyzed overnight against a buffer solution (20 mMBis-tris-HCl buffer solution (pH 7.2), 1 mM EDTA, and 5 mM MgCl₂).

Then, the dialyzed crude enzyme solution was purified by columnchromatography (adsorption: a buffer solution A (20 mM Bis-tris-HClbuffer solution (pH 7.2), 1 mM EDTA, and 5 mM MgCl2), elution: a buffersolution A—0.0 to 0.6 M sodium chloride gradient) using Q-SEPHAROSE FF(manufactured by Amersham Pharmacia Biotech). The Ddl active fractionswere collected and further purified by gel filtration columnchromatography with SEPHACRYL S-100 (manufactured by Amersham PharmaciaBiotech). The obtained active fractions were subjected to SDS-polyacrylamide gel electrophoresis and stained by Coomassie brilliant blue, whichconfirmed that the DdlB was purified to substantially a single band.

EXAMPLE 3 Preparation of Recombinant D-serine Dehydratase (Dsd) Derivedfrom E. coli

(3-1) Preparation of expression vector and transformant including dsdgene

The following two synthetic primers (SEQ ID NOS: 5 and 6) weresynthesized, based on the base sequence information of the dsd gene ofE. coli encoding an enzyme having the Dsd activity.

PCR was performed, using these primers and using genome DNA of E. colias a template. The obtained DNA fragment was digested with EcoRI andHindIII, and ligated to a vector PUC118 that had been digested with thesame restriction enzymes. This was introduced into E. coli JM109 by therubidium chloride method to give a transformant having a recombinantplasmid containing the dsd gene.

(3-2) Preparation of Dsd

The Dsd expressing cell strain obtained in the above-described mannerwas cultured in about 10 L of LB medium containing 100 μg/ml ofampicillin. Enzyme purification was performed according to the methoddescribed in J. Biol. Chem., 263, 16926–16933, 1988. The cell paste wassuspended in SPE lysis buffer (20% sucrose, 20 mM EDTA, 30 mM potassiumphosphate, 0.5 mg/mL lysozyme, pH7.8) in almost the same volume. Afterincubation, water containing a protease inhibitor and pyridoxal5′-phosphorate (PLP) was added thereto for lysis. The lysate was cooledwith ice and a buffer solution B (1M potassium phosphate, 800 μM PLP, 50mM EDTA, 10 mM DTT, pH7.5) was added thereto in almost the same volume,and the cell debris was removed by centrifugation at 18000 rpm for 30minutes. The obtained supernatant was adjusted to pH 7.3, and nucleicacid was precipitated by 1% Polymin P and then centrifuged to givesupernatant. Ammonium sulfate was added thereto and dissolved therein soas to be 70% saturation. Then, the solution was stirred for 40 minutesand then centrifuged at 18000 rpm for two hours. The obtainedprecipitate was suspended in a small amount of buffer solution C (10 mMpotassium phosphate, 80 μM PLP, 1 mM EDTA, 1 mM DTT, pH 7.2) anddialyzed overnight against the same buffer solution.

Then, the dialyzed crude enzyme solution was purified by columnchromatography using DEAE-TOYOPEARL (manufactured by Tosoh Corporation).For adsorption, the buffer solution C was used, and elution wasperformed using 0 to 200 mM KCl gradient in this buffer solution. TheDsd active fractions were collected and precipitated with 70% saturatedammonium sulfate and recovered. The recovered fractions were dissolvedin a small amount of the buffer solution C, and dialyzed overnightagainst the same buffer solution to remove ammonium sulfate.Furthermore, they were dialyzed against a buffer solution D (1 mMpotassium phosphate, 1 mM DTT, pH7.0) for 3 to 4 hours.

The dialyzed partially purified enzyme solution was further purified byhydroxyapatite column chromatography (Gigapite, manufactured by TOAGOSEICO., LTD., available from SEIKAGAKU CORPORATION). This enzyme solutionwas applied to a column previously equilibrated with the buffer solutionD, washed with the same buffer solution in a volume three times thecolumn, and eluted with a buffer solution E (10 mM potassium phosphate,80 μm PLP, 1 mM EDTA, pH7.8). A buffer solution in a volume of 1/10 wasadded to each eluted fraction, and Dsd active fractions were collected,precipitated with 70% saturated ammonium sulfate and recovered. Theobtained precipitate was suspended in a small amount of buffer solutionF (100 mM potassium phosphate, 80 μm PLP, 1 mM EDTA, 1 mM DTT, pH7.8),and dialyzed against this buffer solution to give a purified enzyme.

(3-3) Measurement of Dsd Activity

The Dsd activity was determined by measuring coupling of the pyruvicacid produced from D-serine with lactate dehydrogenase in the presenceof NADH. More specifically, 0.01 to 0.1 U Dsd was added to 100 mMD-serine, 0.5 mM NADH and 5 U lactate dehydrogenase at 37° C. toinitiate a reaction, and a decrease of the absorbance at 340 nm wastraced.

EXAMPLE 4 Prevention of Influence of D-alanine by DdlA, DdlB and Dsd

Samples (1 to 5) were prepared in the following manner:

-   -   Sample 1: Normal control blood serum SERACLEAR HE (manufactured        by Azwell Co. Ltd.)    -   Sample 2: Addition of 25 μM of homocystine (corresponding to 50        μM of homocysteine) to the sample 1    -   Sample 3: Addition of 100 μM of D-alanine to the sample 2    -   Sample 4: Addition of 500 μM of D-serine to the sample 2    -   Sample 5: Addition of 100 μM of D-alanine and 500 μM of D-serine        to the sample 2

First reagents (I to V) and a second reagent were prepared in thefollowing manner:

First Reagent:

-   -   Reagent I: 50 mM Bicine (pH 8.0), 123 U/L homocysteine        methyltransferase (derived from bacteria), 5.6 mM        dithiothreitol, 0.06 mM D-methionine methylsulfonium, 1 mM zinc        bromide, 0.3 mM DA-67, 1 mM ATP, 1 mM magnesium chloride    -   Reagent II: Addition of 0.2285 mg protein/mL of DdlB to the        reagent I    -   Reagent III: Addition of 2.0875mg protein/mL of DdlA to the        reagent I    -   Reagent IV: Addition of 1 U/mL of Dsd to the reagent I    -   Reagent V: Addition of 0.2285 mg protein/mL of DdlB and 1U/mL of        Dsd to the reagent I at the same time

Second Reagent:

-   -   A reagent containing 50 mM citric acid (pH 5.6), 23 mM NEM, 6.4        U/mL D-amino acid oxidase derived from porcine kidney, and 5.5        U/mL peroxidase

Measurement was performed using Hitachi 7170 in the following manner.Any one of the first reagents in an amount of 180 μL was added to eachof the samples 1 to 5 in an amount of 15 μL, and allowed to react at 37°C. for 5 minutes. Then, 120 μL of the second reagent was added thereto,and further allowed to react at 37° C. for 5 minutes. The absorbancechange (dominant wavelength 660 nm, secondary wavelength 750 nm) fromthe detection point 16th to 34th was measured.

First, the absorbance change for the samples 1 and 2 were measured withthe reagents I to V, respectively, and the measurement sensitivity withrespect to the homocysteine added to the samples in each case was takenas 100%. Then, the absorbance change for the samples 3 to 5 weremeasured with the reagents I to V. The measurement sensitivities of thesamples 3 to 5 were shown in Table 1.

TABLE 1 Sample 5 Sample 3 Sample 4 D-Ala + D-Ala D-Ser D-Ser Reagent IControl 148% 123% 169% Reagent II D-alanyl-D-alanine 112% 119% 128%ligase (B) (DdlB) Reagent D-alanyl-D-alanine 103% 117% 117% III ligase(A) (DdlA) Reagent D-serine 148% 100% 147% IV dehydratase (Dsd) ReagentV DdlB + Dsd 112% 101% 112%

As seen from these results, in the control, because of the influence ofD-alanine and D-serine, the measurement value was high, whereas whenDdlB or DdlA was contained in the measurement reagent, the influence ofD-alanine in the sample was reduced, and when Dsd was contained, theinfluence of D-serine in the sample was reduced. Furthermore, it isevident that when both are used simultaneously, the influence ofD-alanine and D-serine can be reduced at the same time.

EXAMPLE 5 Elimination of D-alanine and D-serine by Ddl and Dsd

Samples (four types) were prepared in the following manner:

-   -   Sample 1: Normal control serum SERACLEAR HE (manufactured by        Azwell Co. Ltd.)    -   Sample 2: Addition of 25 μM of homocystine (corresponding to 50        μM of homocysteine) to the sample 1.    -   Sample 6: Addition of 200 μM of D-alanine to the sample 2    -   Sample 7: Addition of 1000 μM of D-serine to the sample 2

First reagents and a second reagent were prepared in the followingmanner:

First Reagents:

-   -   A reagent in which 0, 0.073, 0.145, 0.29 or 0.58 U/mL of DdlB or        0, 0.125, 0.25 or 0.5 U/ml of Dsd was added to a reagent        containing 50 mM Bicine (pH 8.0), 123 U/L homocysteine        methyltransferase (derived from bacteria), 5.6 mM        dithiothreitol, 0.06 mM D-methionine methylsulfonium, 1 mM zinc        bromide, 0.3 mM DA-67, 5 mM ATP, and 10 mM magnesium chloride

Second Reagent:

-   -   A reagent containing 50 mM citric acid (pH 5.6), 23 mM NEM, 6.4        U/mL D-amino acid oxidase derived from porcine kidney, and 5.5        U/mL peroxidase

Measurement was performed using Hitachi 7170 in the following manner.First, 180 μL of first reagent was added to 15 μL of the sample, andallowed to react at 37° C. for 5 minutes. Then, 120 μL of the secondreagent was added thereto, and further allowed to react at 37° C. for 5minutes. The absorbance change (dominant wavelength 660 nm, secondarywavelength 750 nm) from the detection point 16th to 34th was measured.

The results are shown in FIGS. 4 and 5.

FIG. 4 shows the concentration of DdlB on the horizontal axis, and therelative sensitivity at the time of homocysteine measurement on thevertical axis. It was confirmed that, by using about 0.5 U/mL of DdlB,the influence of 200 mM D-alanine was substantially prevented.

FIG. 5 shows the concentration of Dsd on the horizontal axis, and therelative sensitivity at the time of homocysteine measurement on thevertical axis. By using about 0.2 U/mL of Dsd, the influence of 1000 mMD-serine was substantially prevented.

EXAMPLE 6 Prevention of Influence of D-amino Acid in a Sample by Ddl andDsd

Twelve samples of EDTA plasma were used as samples. As the standard, asample in which 50 μM D-methionine was added to a control blood serumwas used.

Three first reagents (i to iii) and a second reagent (used in common)were prepared in the following manner:

First Reagents:

-   -   Reagent i: 50 mM Bicine (pH 8.0), 126 U/L homocysteine        methyltransferase (derived from bacteria), 5.6 mM        dithiothreitol, 0.06 mM D-methionine methylsulfonium, 1 mM zinc        bromide, 0.3 mM DA-67    -   Reagent ii: excluding the homocysteine methyltransferase from        the reagent i    -   Reagent iii: Addition of 5 mM ATP, 10 mM magnesium chloride,        0.58 U/mL DdlB, and 1 U/mL Dsd to the reagent i

Second Reagent:

-   -   A reagent containing 50 mM citric acid (pH 5.6), 23 mM NEM, 6.4        U/mL D-amino acid oxidase derived from porcine kidney, and 5.5        U/mL peroxidase

Measurement was performed using Hitachi 7170 in the following manner.Any one of the first reagents in an amount of 180 μL was added to 15 μLof the sample, and allowed to react at 37° C. for 5 minutes. Then, 120μL of the second reagent was added thereto, and further allowed to reactat 37° C. for 5 minutes. The absorbance change (dominant wavelength 660nm, secondary wavelength 750 nm) from the detection point 16th to 34thwas measured. The homocysteine concentration in the sample was obtainedbased on the absorbance change of the standard. The value obtained as aresult of measurement with only the reagent A was taken as the valueaccording to the conventional single channel method, the value obtainedas a result of taking a difference between measurements with the reagenti and the reagent ii was taken as the value according to theconventional double channel method, and the value obtained as a resultof measurement with the reagent iii was taken as the value according tothe method of the present invention. Each of these values was comparedwith the value measured according to the HPLC method.

As seen from FIG. 6, compared with the conventional single channelmethod, the method of the present invention has an improved correlationwith the HPLC method, and exhibits a comparable correlation to theconventional double channel method.

1. A method for detecting or measuring homocysteine in a sample,comprising: a) reacting a D-amino acid present in a sample with aD-amino acid converting enzyme to convert the D-amino acid into asubstance that does not serve as a substrate of D-amino acid oxidase orD-amino acid acetyltransferase; (b) reducing homocysteine in the samplewith a thiol compound; (c) reacting the reduced homocysteine with amethyltransferase and a methyl donor to newly produce a D-amino acid;and (d) reacting the produced D-amino acid with the D-amino acid oxidaseor the D-amino acid acetyltransferase in the presence of an SH reagentto produce hydrogen peroxide, and color-developing the produced hydrogenperoxide by using an oxidative color-developing agent.
 2. The method ofclaim 1, wherein the step (a) is a step of reacting D-alanine present ina sample with D-alanyl-D-alanine ligase in the presence of adenosinetriphosphate to convert the D-alanine into D-alanyl-D-alanine and/or astep of reacting D-serine present in a sample with D-serine dehydrataseto convert the D-serine into pyruvic acid.
 3. The method of claim 2,wherein the methyltransferase is homocysteine methyltransferase, and themethyl donor is D-methionine methylsulfonium.
 4. The method of claim 3,wherein in the step (d), the produced hydrogen peroxide is detected ormeasured by color-development using peroxidase and an oxidativecolor-developing agent.
 5. The method of claim 2, wherein in the step(d), the produced hydrogen peroxide is detected or measured bycolor-development using peroxidase and an oxidative color-developingagent.
 6. A reagent kit for measuring homocysteine comprisingD-alanyl-D-alanine ligase and/or D-serine dehydratase; a thiol compound;a methyltransferase; a methyl donor; D-amino acid oxidase or D-aminoacid acetyltransferase; an SH reagent; and an oxidative color-developingagent.